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Biomedical subjects

A Rebollo

Publications and source records attributed to A Rebollo.

At least 19 recordsLinked to original sources
PubMed2006

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers.

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.

Biosensing Techniques↗
PubMed2005

Association between bone mineral density and polymorphisms of the VDR, ERalpha, COL1A1 and CTR genes in Spanish postmenopausal women.

Bone mineral density (BMD), the major determinant of osteoporotic fracture risk, has a strong genetic component, and several candidate gene polymorphisms have been implicated in the regulation of this process. In view of the reported associations between the BMD and polymorphisms in the collagen type I alpha 1 gene (COL1A1), vitamin D receptor (VDR), estrogen receptor (ER)alpha and calcitonin receptor (CTR) genes, an association study was performed between VDR, COL1A1, CTR and ER genotypes and lumbar spine, femoral neck and Ward's triangle BMD in postmenopausal Spanish women. We statistically controlled for many confounding factors, such as height, weight, age, years since menopause, use of hormone replacement therapy (HRT), tobacco consumption, use of oral contraceptives, calcium dietary intake or exercise practice. No association between COL1A1 or ER genotypes and BMD was detected. However, we described a statistically significant association between a personal history of fractures and COL1A1 genotype. The ss genotype was found to be over-represented between those women who had a personal history of fractures. The analyses of the VDR polymorphisms showed that FF subjects reached the highest values of BMD at the three measured sites, whereas Ff individuals had an intermediate BMD and ff women had the lowest values. However, the VDR-BsmI gene polymorphism was not found to be associated with adjusted BMD. For the CTR polymorphisms, our study showed that women with the aa genotype had a lower adjusted BMD at the femoral neck. In conclusion, in our postmenopausal Spanish women cohort we found a statistically significant association beteween the VDR and CTR gene polymorphisms and the BMD. However, we did not find any association between the ER and COL1A1 gene and the BMD. The COL1A1 gene was found to be associated with the prevalence of osteoporotic fractures. Of all the studied gene polymorphisms, the FokI VDR gene polymorphism seems to be the strongest BMD genetic determinant of postmenopausal Spanish women.

Adult↗
PubMed2002

Aiolos and Ikaros: regulators of lymphocyte development, homeostasis and lymphoproliferation.

Aiolos and Ikaros encode hemopoietic-specific zinc finger transcription factors that are important regulators of lymphocyte differentiation. Aiolos and Ikaros play a critical role in regulating B and T cell development. Gene targeting studies in mice have shown that inactivation of Ikaros family proteins leads to a complete absence of T, B, NK and dendritic cells, whereas a reduction of Ikaros activity induce hyperproliferation and lymphomas. Aiolos knock-out mice have quantitatively normal lymphoid cells but have chronically activated B cells producing autoantibodies and develop lymphomas with increased frequency. These proteins are involved in the control of gene expression and, associated to nuclear complexes, participate in nucleosome remodeling. This protein family governs cell fate decisions and regulates homeostasis through complex isoforms expression and dimerization. Changes in this regulatory network may reflect differentiation and proliferation adjustments made in lymphoid progenitors and precursors. The direct involvement of aberrant Ikaros protein expression in human hematological oncogenesis, although suggested by several studies, remains to be settled at the genomic level. These points will be discussed in the present review.

Amino Acid Sequence↗
PubMed2001

The association of Aiolos transcription factor and Bcl-xL is involved in the control of apoptosis.

We have analyzed the mechanism implicated in the control of the anti-apoptotic role of Bcl-xL. We show that IL-4 deprivation induces apoptosis, but does not modulate Bcl-xL expression. Because Bcl-xL does not promote cell survival in the absence of IL-4, we investigate the mechanism by which Bcl-xL was unable to inhibit apoptosis. Using yeast two-hybrid system, coimmunoprecipitation, and indirect immunofluorescence techniques, we found that Bcl-xL interacts with the transcription factor Aiolos in IL-4-stimulated cells, increasing upon IL-4 deprivation. IL-4 does not promote translocation of Aiolos or Bcl-xL, but induces tyrosine phosphorylation of Aiolos, which is required for dissociation from Bcl-xL. Transfection experiments confirm that cells overexpressing Bcl-xL are able to prevent apoptosis in the absence of IL-4. On the contrary, cells that overexpress Bcl-xL and Aiolos are unable to block apoptosis in the absence of IL-4. We propose a model for the regulation of the Bcl-xL anti-apoptotic role via Aiolos.

Animals↗
PubMed2001

Bcl-2 targets protein phosphatase 1 alpha to Bad.

The diverse forms of protein phosphatase 1 (PP1) in vivo result from the association of the catalytic subunit with different regulatory subunits. We recently have described that PP1alpha is a Ras-activated Bad phosphatase that regulates IL-2 deprivation-induced apoptosis. With the yeast two-hybrid system, GST fusion proteins, indirect immunofluorescence, and coimmunoprecipitation, we found that Bcl-2 interacts with PP1alpha and Bad. In contrast, Bad did not interact with 14-3-3 protein. Bcl-2 depletion decreased phosphatase activity and association of PP1alpha to Bad. Bcl-2 contains the RIVAF motif, analogous to the well characterized R/KXV/IXF consensus motif shared by most PP1-interacting proteins. This sequence is involved in the binding of Bcl-2 to PP1alpha. Disruption of Bcl-2/PP1alpha association strongly decreased Bcl-2 and Bad-associated phosphatase activity and formation of the trimolecular complex. These results suggest that Bcl-2 targets PP1alpha to Bad.

Amino Acid Sequence↗
PubMed2000

Protein phosphatase 1alpha is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis.

Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1alpha (PP1alpha). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo (32)P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1alpha. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras.

Animals↗
PubMed2000

Bcl-3 expression promotes cell survival following interleukin-4 deprivation and is controlled by AP1 and AP1-like transcription factors.

We have analyzed the interleukin-4 (IL-4)-triggered mechanisms implicated in cell survival and show here that IL-4 deprivation induces apoptotic cell death but does not modulate Bcl-2 or Bcl-x expression. Since Bcl-x expression is insufficient to ensure cell survival in the absence of IL-4, we speculate that additional molecules replace the antiapoptotic role of Bcl-2 and Bcl-x in an alternative IL-4-triggered pathway. Cell death is associated with Bcl-3 downregulation and Bcl-3 expression blocks IL-4 deprivation-induced apoptosis, suggesting that Bcl-3 acts as a survival factor in the absence of growth factor. To characterize the IL-4-induced regulation of murine Bcl-3 expression, we cloned the promoter of this gene. Sequencing of the promoter showed no TATA box element but did reveal binding sites for AP1, AP1-like, and SP1 transcription factors. Retardation gels showed that IL-4 specifically induces AP1 and AP1-like binding activity and that mutation of these binding sites abolishes the IL-4-induced Bcl-3 promoter activity, suggesting that these transcription factors are important in Bcl-3 promoter transactivation. IL-4 deprivation induces downregulation of Jun expression and upregulation of Fos expression, both of which are proteins involved in the formation of AP1 and AP1-like transcription factors. Overexpression of Jun family proteins transactivates the promoter and restores Bcl-3 expression in the absence of IL-4 stimulation. Taken together, these data describe a new biological role for Bcl-3 and define the regulatory pathway implicated in Bcl-3 expression.

Animals↗
PubMed1999

Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis.

IL-2 deprivation triggers apoptosis in the murine T cell line TS1alphabeta, a process that can be blocked by overexpression of Bcl-2. Here we show that Bcl-2 and Ras proteins interact in mitochondria from TS1alphabeta cells in the presence or absence of IL-2, as evidenced by co-immunoprecipitation. All three Ras proteins, K-, N- and H-Ras, interact with Bcl-2; however, their mitochondrial localization is differentially regulated in IL-2-supplemented or -deprived cells. K-Ras is found in mitochondria only in IL-2-supplemented cells, whereas H-Ras is observed in mitochondria only after IL-2 withdrawal. N-Ras is detected in mitochondria under both experimental conditions. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras posttranslational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. These results evidence a distinct behavior among the three Ras proteins in TS1alphabeta cells, depending on IL-2 supply, and suggest homologue-specific roles for Ras proteins in IL-2-dependent events.

Animals↗
PubMed1999

Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization.

We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos.

Animals↗
PubMed1999

IL-2 deprivation triggers apoptosis which is mediated by c-Jun N-terminal kinase 1 activation and prevented by Bcl-2.

A variety of environmental stresses, as well as inflammatory cytokines, induce activation of c-Jun N-terminal kinases. We describe here that IL-2 deprivation-induced apoptosis in TS1alphabeta cells does not modify c-Jun protein levels and correlates Bcl-2 downregulation and an increase in JNK1, but not JNK2, activity directly related to the induction of apoptosis. Indeed, downregulation of JNK1 expression using antisense oligonucleotides inhibits apoptosis induced by IL-2 withdrawal. Overexpression of Bcl-2 promotes cell survival and blocks JNK1 activation as well as apoptosis caused by IL-2 deprivation. This suggests that inhibition of the JNK1 signaling pathway may be a mechanism through which Bcl-2 promotes cell survival and prevents apoptosis triggered by growth factor withdrawal.

Animals↗
PubMed1999

Novel aspects of Ras proteins biology: regulation and implications.

The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.

Animals↗
PubMed1999

[CEA, CA 19.9 and CA 195 in patients with colorectal carcinoma. ROC analysis].

The aim of this study was to assess the utility of determining serum levels of the CEA, CA 19.9 and CA 195 tumor markers in 906 patients with colorectal carcinoma for a 5-year follow-up period. In addition to calculating sensitivity and specificity for the common normal limits, the results were analyzed with RO curves. Sensitivity values for 90% and 95% specificity were also obtained. The results of the areas under the curve (CEA: 0.887; CA 19.9: 0.802; CA 195: 0.971) and sensitivities and specificities at different normality limits showed better behavior for CEA with respect to CA 19.9 and CA 195, the last two presenting similar results. CEA continues to be the best tumor marker to monitor patients with colorectal carcinoma.

Adenocarcinoma↗
PubMed1998

The Bcl-2 gene is differentially regulated by IL-2 and IL-4: role of the transcription factor NF-AT.

The murine TS1alphabeta T cell line expresses the anti-apoptotic protein Bcl-2 upon IL-2 stimulation, whereas IL-4-mediated growth of this cell line proceeds in the absence of Bcl-2 expression. In addition, IL-4 stimulation inhibits Bcl-2 expression and modulates its mRNA level. IL-2-induced DNA binding activity for these transcription factors is sensitive to phosphatidylinositol 3 kinase inhibitor wortmannin and to Rho inhibitor Clostridium difficile toxin B, which inhibit IL-2-induced Bcl-2 expression. NF-AT transcription factor appears to be the most important in the control Bcl-2 expression, since inhibition of the calcium-calmodulin-dependent phosphatase calcineurin, which regulates NF-AT activity, downregulates Bcl-2 expression in IL-2-stimulated cells. Constitutive expression of this phosphatase also upregulates Bcl-2 expression in IL-4-stimulated cells. In addition, a dominant negative NF-AT expression vector downregulates Bcl-2 expression in IL-2-stimulated cells. These results suggest that IL-2 induction of Bcl-2 expression may be directly or indirectly mediated by NF-AT.

Androstadienes↗
PubMed1998

Dual role of Ras and Rho proteins: at the cutting edge of life and death.

Small GTP-binding proteins of the Ras superfamily are master controllers of the cell physiology. The range of processes in which these proteins are involved include cell cycle progression, cell division, regulation of cell morphology and motility and intracellular trafficking of molecules and organelles. The study of apoptosis, the physiological form of cell suicide, is progressively linking the functions of small G proteins to the control of the mechanisms that trigger the genetic programmes of cell death. To date, isoforms of the Ras and Rho groups have been related to both promotion and suppression of apoptosis. Further, signalling pathways driven by these proteins have been associated with the function and/or expression of molecules that regulate apoptotic responses. Thus, all available evidence points to a critical role for Ras and Rho proteins as major gatekeepers of the decision between cellular life and death.

Apoptosis↗
PubMed1998

Role of Akt and c-Jun N-terminal kinase 2 in apoptosis induced by interleukin-4 deprivation.

We have shown previously that interleukin-4 (IL-4) protects TS1alphabeta cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1alphabeta cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1alphabeta survival is independent of Bcl-2, Bcl-x, or Bax.

Animals↗
PubMed1998

IL-2-induced cellular events.

In this review we discuss several molecules that are attractive candidates as transducing molecules involved in signaling processes. IL-2 receptor signaling is a complex process involving a large number of molecules: Ras, Rho, PI3 kinase, PKC, Akt, transcription factors NF-AT, and NF-kappaB and some target genes such as bcl-2, c-myc, c-jun and c-fos. Ras and Rho have been defined as dual molecules because Ras- and Rho-initiated signals can either promote or inhibit apoptosis. Several studies have contributed to the delineation of a signaling pathway structured in three independent channels designated channels 1, 2, and 3. These three channels serve as major landmarks: Lck-c-fos/c-jun (channel 1), Syk-myc (channel 2), and a pathway leading to actin organization/bcl-2 expression (channel 3). The detailed hierarchical organization of these three channels is presented throughout the review and the model is depicted in the figure.

Animals↗
PubMed1997

Role of NF-kappaB in the control of apoptotic and proliferative responses in IL-2-responsive T cells.

The NF-kappaB/Rel/IkappaB family of transcription factors regulates a number of genes involved in a wide variety of biological processes. The activation of p53, c-myc and Ras genes suggests a role for NF-kappaB in cell proliferation; NF-kappaB is also important in immune and inflammatory responses. By virtue of its role in apoptosis, NF-kappaB participates in the thymus as well as in embryonic development. The NF-kappaB family of transcription factors is also involved in viral transcription, transformation and in the development of some types of human cancers. Given the pivotal role of NF-kappaB, clarification is needed of the mechanisms through which its deregulation contributes to disease. Several aspects of NF-kappaB regulation, such as phosphatase involvement, the mechanism of IkappaB ubiquitination and the regulation of nuclear translocation, remain obscure. Here, we review and discuss the function of NF-kappaB activation in IL-2-stimulation and in apoptosis induced by IL-2 deprivation in T cells.

Animals↗